JOURNAL

OF SURGICAL

RESEARCH

48, 373-378 (1990)

Impact of Postantibiotic Effect on Bacterial Adherence to Vascular Prostheses DAVID D. SCHMITT, M.D., CHARLES E. EDMISTON, PH.D., CANDACE KREPEL, B.S., CLAUDIA GOHR, B.S., GARY R. SEABROOK, M.D., DENNIS F. BANDYK, M.D., AND JONATHAN B. TOWNE, M.D. Department

of Surgery and

the Surgical Microbiology Research Laboratory, Medical College

Presented at the Annual Meeting of the Association

for Academic Surgery, Louisville,

The brief exposure of bacteria to high antibiotic concentrations can result in prolonged suppression of bacterial growth termed postantibiotic effect (PAE). A pathogenic Staphylococcus epidermidis strain (RP-62) was exposed to 2X and 6X minimum inhibitory concentrations (MIC) of cefazolin, vancomycin, or rifampin for 1 hr and incubated for 2 to 24 hr in inhibitory-free broth. PAE was defined as the difference in time required for a 1.0 log increase in test (T) vs control (C) cultures (PAE = T - C). PAE was observed only for rifampin: 6 hr at 2X MIC and 8 hr at 6X MIC. Bacterial adherence to Dacron grafts was calculated in PAE vs control cultures by a quantitative culture technique for graft specimens incubated 2 to 24 hr. A demonstrable PAE and its impact on adherence were found to be both antimicrobial and concentration dependent. A significant decrease in staphylococcal adherence to velour-knitted Dacron was demonstrated by rifampin at 2X MIC (P < 0.05) and 6X MIC (P < 0.01). This phenomena may be useful in reducing bacterial adherence and colonization of bioprosthetics in the perioperative period. Preoperative suppression of staphylococcal skin flora by high dose antimicrobials can alter the capacity of these organisms to adhere to vascular prosthetic grafts and, if incorporated into antibiotic prophylaxis regimens, may reduce graft colonization. 0 1990 Academic Press, Inc.

INTRODUCTION

of Wisconsin, Milwaukee,

Kentucky,

15-18,1989

identified as the predominant pathogen in infections of prosthetic vascular grafts and other bioprostheses [6-B]. Disrupting or inhibiting the initial adherence of S. epidermidis to synthetic conduits would have a significant impact on reducing the morbidity and mortality associated with biomaterial implantation. This study examined the capacity of high dose antimicrobials to induce a PAE on a clinical strain of S. epidermidis and to alter adherence to velour-knitted Dacron by a quantitative culture technique. MATERIALS

AND

METHODS

Organism The study strain was S. epidermidis (RP-62) ATCC 35984 (obtained from Dr. Gordon Christensen, University of Missouri, Columbia, MO) and was identified with the STAPH-IDENT system (Analytab Products, Plainview, NY). This strain is catalase-positive and coagulase-negative and sensitive to novobiocin and fermented glucose, but not mannitol. The organism exhibits slime production following overnight growth in glass test tubes containing trypticase soy broth with 0.25% dextrose. This exopolysaccharide is visualized when stained with Alcian blue according to the qualitative method of Christensen et al. [9]. Antibiotics and Minimal Determination

Postantibiotic effect (PAE) is the persistent suppression of bacterial growth after brief exposure to high concentrations of antimicrobials [ 11. This phenomena has been described for gram-positive and gram-negative microorganisms in both in vitro and in uiuo models [2-51. Bacteria that are in a suppressed or “PAE” state may have altered virulence characteristics due to inhibition or alteration of the organism’s cellular mechanisms. Bacterial adherence is the inceptive step in the colonization and subsequent infection of an implanted vascular prosthesis. Staphylococcus epidermidis has been

November

Wisconsin 53226

Inhibitory

Concentration

Minimal inhibitory concentrations (MIC) of the study strain were determined for cefazolin (Smith, Kline and French Laboratories, Philadelphia, PA), vancomycin (Eli Lilly and Co., Indianapolis, IN), and rifampin (Merrell Dow, Cincinnati, OH). Antibiotics were prepared in stock solutions as recommended by the manufacturer and stored at -70°C. MIC were determined by the tube microdilution method using Mueller-Hinton broth (GIBCO Diagnostics, Madison, WI) following the National Committee for Clinical Laboratory Standards [lo]. After 24 hr of incu373

0022-4804/90

$1.50

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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The postantibiotic effect was defined as the difference between the time required for the colony forming units in the test (7’ = time in hours) versus control tubes (C = time in hours) to increase 1.0 log,, above the count observed immediately following drug removal [ 11. Therefore, the PAE (7’ - C) is the time interval during which normal bacterial growth was inhibited after drug removal.

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FIG. 1. Growth kinetics of S. epidermidis (RP-62). Control and test cultures exposed to 2X and 6X MIC of cefazolin for 1 hr. No PAE exhibited.

bation on blood agar, five colonies were inoculated to Mueller-Hinton broth and incubated for 18 hr at 35°C. The inoculum was adjusted to a 0.5 McFarland Standard with Mueller-Hinton broth. The inoculum density in each tube was approximately 6.0 log,, CFU/ml. The tubes were incubated at 35°C for 18-24 hr. The MIC was defined as the lowest concentration of antimicrobial agent that inhibited visual growth of the organism. Determination

of PAE

In vitro PAE was determined by the broth centrifugation method [ 11. The S. epidermidis (RP-62) strain was grown overnight (24 hr) in Mueller-Hinton broth. The overnight broth culture was adjusted spectrophotometrically to an optical density of 0.3 at 580 nm, and 1 ml of the adjusted inoculum was added to a tube containing 9 ml of Mueller-Hinton broth and the test antimicrobial and to a tube containing 9 ml of an inhibitory free broth control. The final microbial concentration in each tube was approximately 7.5 log,, CFU/ml. The antimicrobials were added in test concentrations of 2X and 6X the MIC of the isolate. Test and control cultures were incubated at 35°C. After 1 hr of antibiotic exposure, the broth culture media was centrifuged (1200g for 10 min) and washed (3X) with buffered dilutant to remove the test drug. This step was repeated for the control culture. Drug elimination was confirmed by testing a 200-~1 aliquot from the final wash solution for antimicrobial activity by biologic assay. Following the final wash, antibiotic and control cultures were incubated at 35°C. A lOO-~1 aliquot was removed at 0,l (pre- andpostwash), 2,4,8,12,18, and 24 hr postdrug removal. Dilutions were made in pH 7.4 phosphate-buffered saline (PBS) and plated in duplicate on trypticase soy agar. Plates were read at 24 and 48 hr after incubation at 35°C.

The impact of PAE on staphylococcal adherence to velour-knitted Dacron (VKD) was determined by first preparing control and test inocula exposed to antimicrobials as outlined above. Following the final wash the inocula were adjusted spectrophotometrically to a concentration of approximately 5.7 log,, CFU/ml. Commercially available, VKD graft material (Meadox Medicals, Inc., Oakland, NJ) was used in the staphylococcal studies. Graft material was cut into l-cm squares and sterilized. Graft specimens were immersed in the inoculating solution and incubated at 35°C. Specimens were removed at 2, 4, 8, 12, 18, and 24 hr and washed three times with PBS to remove nonadherent organisms. Three specimens were evaluated for each time interval. Each graft specimen was placed in 40 ml of PBS and sonicated at 20 kHz for 10 min to dislodge adherent organisms from the graft fibers. One milliliter of the sonication effluent was then backplated at 10m3and 10m4dilutions on tryptic soy agar. The quantitative culture plates were read at 24 and 48 hr. Bacterial adherence was calculated by multiplying the number of CFU dislodged from the specimen times 40 (the volume of the sonication effluent). Final staphylococcal adherence was expressed as the number of CFU per square centimeter of graft material [ 111. Data from bacterial adherence studies were compared for statistical significance with the two-sample t test, with P < 0.05 considered significant. 9.4

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SCHMITT

ET AL.: IMPACT

OF POSTANTIBIOTIC

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FIG. 3. Postantibiotic effect for S. epidermidis (RP-62) after 1 hr of exposure to rifampin. PAE = time for test [17 hr (2X MIC) 19 hr (6X MIC)] vs time for control (11 hr) to increase 1.0 log,, CFU/ml. PAE = 6 hr (2X MIC) and 8 hr (6X MIC).

Scanning Electron Microscopy (SEM) Graft specimens for SEM were prefixed for 24 hr in 2% glutaraldehyde buffered in 0.15 M sodium cacodylate (pH 7.4). The specimens were washed twice in buffer followed by postfixation for 24 hr in osmium tetroxide. Following three buffered rinses, the graft material was serially dehydrated in ethanol, critical point dried from liquid carbon dioxide, and coated with gold palladium. The grafts were viewed in a Phillips 500 scanning electron microscope at 25 kV and a spot size of 8 nm. RESULTS

The MIC for S. epidermidis strain RP-62 was 8.0 pg/ ml for cefazolin, 2.0 pg/ml for vancomycin, and 0.5 pg/ ml for rifampin. The growth kinetics following 1 hr of exposure to 2X and 6X MIC concentration of cefazolin, vancomycin, and rifampin was calculated as the mean values of three graft specimens for each time interval (Figs. l-3). No postantibiotic effect was demonstrated for cefazolin or vancomycin with exposure at either 2X or 6X the MIC when compared to the control cultures. However, following 1 hr of incubation with rifampin, at 2X MIC 17 hr was required, and at 6X MIC 19 hr was required, to achieve a 1.0 log,, growth while control organisms achieved this level of growth in only 11 hr. Therefore, PAE (T - C) was 6 hr for 2X MIC and 8 hr for 6X MIC (Fig. 3). Figure 4 depicts the mean quantitative microbial adherence of S. epidermidis RP-62A to the Dacron graft material for control and rifampin PAE-suppressed organisms. During the 24 hr incubation period, the number of organisms in the inoculum effluent did not change significantly. The impact of rifampin-induced PAE resulted

375

EFFECT

in a significant decrease in staphylococcal adherence at 2X and 6X the MIC. Furthermore, the 6X MIC-exposed staphylococcal cells demonstrated a greater decrease in adherence which was more persistent than that of the 2X MIC-exposed cells. The 2X MIC-exposed microorganisms demonstrated decreased adherence for 12 hr following antibiotic exposure (P < 0.05) as compared to that of control organisms, while the 6X MIC rifampin-exposed culture demonstrated a persistent suppression throughout the 24-hr adherence assay (P < 0.01) compared to the antibiotic free controls. Exposure to cefazolin and vancomycin had no effect on staphylococcal adherence to the VKD graft material. The impact of high dose antibiotic exposure on adherence to the prosthetic graft material can be seen by scanning electron microscopy. The grafts infected with control staphylococcal organisms demonstrate the formation of large vegetative microcolonies on the prosthetic surface (Fig. 5A), while staphylococci exposed to 6X MIC of rifampin colonized the graft fibers as single adherent organisms rather than microcolonial clusters compared to control graft cultures (Fig. 5B). DISCUSSION

The suppression of bacterial growth following short exposure to antimicrobials was first recognized over 40 years ago. Bigger [ 121, followed later by Parker and Marsh [ 131, observed delayed staphylococcal and streptococcal growth after brief exposure to penicillin G. More recently, the term PAE has been used by Craig and Gundmundsson to describe this period of persistent suppression of bacterial growth, emphasizing that the effect is due to prior

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FIG. 4. Quantitative bacterial adherence of S. epidermidis (RP-62) to velour-knitted Dacron. Data are log,, CFU/cm’ following exposure of graft material to control, 2X MIC and 6X MIC of rifampin at time intervals of 2 to 24 hr (n = 3). Statistical significance by two-sample t test: I’ < 0.05 for 2X MIC and P < 0.01 for 6X MIC of rifampin.

376

FIG. 5. demonstrate

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1990

Scanning electron micrografts of S. epidermidis control (A) and test 6X MIC-rifampin exposed (B) organisms. Control large microcolony formation, while antibiotic-exposed bacteria adhere as isolated organisms to the graft fibers (magnification

antibiotic exposure rather than to the presence of subinhibitory serum or tissue concentrations of the drug [ 11. The present data suggest that there may be an application of PAE in the prophylactic use of antibiotics at the time of prosthetic vascular graft or other biomaterial implantation. The antibiotics studied were chosen because they are commonly used in the clinical treatment of established coagulase-negative infection. Each of these antibiotics act by a different mechanism. Cefazolin interferes in the cell wall synthesis; vancomycin inhibits peptidoglycan formation; rifampin inactivates DNA-dependent RNA polymerase. While no PAE or effect on adherence characteristics was observed with either cefazolin or vancomycin, exposure to rifampin resulted in both a PAE and a significant decrease in adherence as compared to controls. The exact mechanisms by which antimicrobials are able to induce postantibiotic suppression of growth have not been determined. The ,&lactam antibiotics inactivate the penicillin binding proteins by acylating their active sites when susceptible organisms are exposed to these agents. Following removal of the bacteria from the antibioticcontaining medium, several processes may occur before normal growth resumes. There may be a recovery of druginactivated enzyme molecules, reversible damage to the cell wall structures may be repaired, or there may be a synthesis of new enzymes which mediate cell wall septa

bacteri 3200X

formation [ 141. Similarly, the PAE observed with agents which interfere with protein or RNA synthesis may be due to an alteration of regulatory proteins necessary for the organism’s intermediary metabolism and uninterrupted growth patterns [ 11. The major clinical significance of the PAE is its application in the timing of antibiotic dosing regimens. The presence of a PAE could be used in designing intermittent or continuous antibiotic dosing schedules. The demonstration of a prolonged PAE suggests that antimicrobial serum and tissue levels could fall below the MIC and still be effective due to the persistent inhibitory action on the bacteria. However, the absence of a PAE may require continuous or more frequent dosing to obtain maximal antibiotic efficacy. The administration of a short course of high dose antibiotics could induce a state of decreased microbial virulence through impaired adherence, diminished tissue invasion, or inhibited toxin release. The current reported incidence of vascular graft infections is between 1 and 3% with a significant associated morbidity and mortality. Despite the routine use of perioperative antibiotics for patients undergoing vascular prosthetic reconstructions, biomaterial infections continue to occur [15-171. Antibiotic dosing regimens are based on clinical data obtained from the evaluation of surgical wound infections, rather than from studies of actual prosthetic infections. Antibiotic prophylaxis for

SCHMITT

ET AL.: IMPACT

wound infections may not be applicable for patients in whom prosthetic material is being implanted. The presence of a foreign body alters the pathogenesis of the infectious process. Both Escherichia coli and 5’. aureus will produce clinical infections in the presence of a prosthetic in inocula concentrations several orders of magnitude less than when no prosthetic material is present. S. epidermidis appears to be even more dependent on the presence of a foreign body to produce infection. Christensen et al. found a plastic catheter was necessary for the development of a S. epidermidis infection, even in the presence of a large microbial challenge (9.0 log,, CFU/ml) [18]. The presence of a foreign body interferes with the host’s clearance of these normally nonvirulent organisms. S. epidermidis is now recognized as the most common organism responsible for infection of implanted prosthetic graft materials [19, 201. Adherence of organisms to the prosthetic surface probably occurs at the time of implantation by bacteria present on the cut surface of the skin, within contaminated lymphatics, or present on the arterial wall or luminal thrombus [21, 221. Once adherent to the graft surface, the staphylococci become difficult to eradicate from the prosthetic [ 23,241. We have demonstrated that bacteria in a PAE-suppressed state have altered adherence characteristics persisting for hours after short, high dose antimicrobial exposure. Theoretically, pretreating a patient with high dose antibiotics prior to implantation of a prosthetic device would produce tissue and serum levels sufficient to induce a PAE on the host’s endogenous flora at the time of surgery resulting in a diminished capacity to colonize a vascular prosthesis. The demonstration of in vitro PAE has been widely used to evaluate the response of both gram-negative and gram-positive bacteria to many antimicrobials. However, less conclusive data have been collected for in uiuo PAE suppression. Two animal models currently exist (a thigh infection in mice and a meningitis model in rabbits) for the in viuo evaluation of PAE [25, 261. Unfortunately, both of these models fail to demonstrate a good correlation between in vitro and in uiuo results relative to clinical outcome. Furthermore, these models deal primarily with treatment of infections and not with prophylaxis. Livingston et al. have recently shown in a rat wound infection model that high dose antibiotic regimens given over both short and long courses of administration were superior to standard doses of the same duration [27]. Although they did not evaluate microbial growth rates, an induced PAE may explain the difference between their experimental groups. Further investigation of PAE suppression by prophylactic antibiotic administration will require the development of an animal model which correlates with in vitro data. The therapeutic application of PAE may become an important future consideration for the prophylactic administration of antibiotics when a prosthetic material is to be implanted.

OF POSTANTIBIOTIC

EFFECT

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Impact of postantibiotic effect on bacterial adherence to vascular prostheses.

The brief exposure of bacteria to high antibiotic concentrations can result in prolonged suppression of bacterial growth termed postantibiotic effect ...
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